The goal of this project is to develop mariner transposable elements as genetic tools for arthropods, primarily insects, of medical importance. The impact of arthropod vectors of disease around the world is enormous, and novel approaches to their control are needed. These mariner transposons are potentially useful for gene tagging for cloning, for enhancer trapping, and most importantly as transformation vectors, both for basic research and practical approaches to mitigating transmission of arthropod-borne diseases. There is an extraordinary diversity of mariner family transposons in the genomes of diverse animals. They appear to function independently of host specific factors, and therefore any particular mariner transposon is a candidate for development. A particular element called Himar1 has been extensively studied by our laboratory, and shown to be active in vitro and in vivo in Drosophila melanogaster. This activity is, however, limited by a concentration- dependent inhibition of the mariner transposase, which appears to be a general feature of mariner transposons and may be shared with some bacterial transposons such as Tn5. We propose to overcome this inhibition by isolating hyperactive uninhibited versions of Himar1 using a mutagenic screen in bacteria, such as E. coli. These will be tested in vitro and then in vivo in D. melanogaster, where they will be developed into transformation vectors, followed by distribution to colleagues for testing in diverse insects of medical importance. Additional hyperactive versions of Himar1 might be obtained by selecting for changes in their termini and flanking DNA. The fate of Himar1 transposase within insect cells and embryos will be examined to determine whether it can be stabilized. The interactions of Himar1 transposase with other mariners of diverse subfamilies will be examined to evaluate whether Himar1 will interact with endogenous mariners in host genomes. This project will hopefully lead to the availability of an efficient general transformation system for diverse arthropods of medical, economical, and basic research importance, and may also lead to development of genetic tools in other organisms.
